In vivo occurrence of carbonyl residues in Phaseolus vulgaris proteins as a direct consequence of a chronic ozone stress

We aimed to show that a chronic and realistic ozone stress could induce in vivo formation of carbonyl groups in leaf proteins of bean (Phaseolus vulgaris L. cv Bergamo). Plants were grown in three open-top chambers with increasing ozone concentrations: non-filtered air (NF), NF+40 nL·L^–1, NF+80 nL·L^–1 ozone 7 h·d^–1 for 22 d. Carbonyl contents in proteins, evaluation of Rubisco (EC 4.1.1.39) amounts and visible damages were systematically investigated in primary and first trifoliate leaves. Visible ozone injuries clearly reflected the total external ozone dose (expressed as AOT40) that the leaf had suffered. Ozone was effective at inducing aldehydes and ketones formation in bean proteins. This production of carbonyl groups increased with ozone concentration, the most relevant difference being observed on the Rubisco small subunit (Rubisco SSU). Contrary to young first trifoliate leaves, older primary leaves from O₃-enriched atmospheres exhibited a two-fold decrease in Rubisco level. Carbonyl group formation in Rubisco SSU and decrease in Rubisco level were not necessarily linked. Depending on ozone concentration, exposure time and leaf age, these two effects were observed either together or separately for an almost similar external dose of ozone. To conclude, leaf symptoms, loss of Rubisco and oxidized Rubisco SSU could participate in the assessment of the impact of a chronic ozone stress.     

Effect of a chronic and moderate ozone pollution on the phenolic pattern of bean leaves (Phaseolus vulgaris L. cv Nerina): relations with visible injury and biomass production

From sowing, bean (Phaseolus vulgaris L. cv Nerina) plants were exposed to three chronic doses of ozone for 7h.day(-1): non-filtered air (NF), non-filtered air supplied with 40nl.l(-1) ozone (NF+40) and non-filtered air supplied with 60nll(-1) ozone (NF+60). Four harvests were carried out 6, 13, 20 and 27 days after emergence. Either primary leaves, or first trifoliate leaves, or both were sampled as far as possible. For each sampled leaf, visible ozone injuries were registered, the free polyphenolic pool was analysed using HPLC and the dry matter was weighed. Visible damage on leaves was related to both exposure time and ozone concentration added. There were no adverse effects of added ozone on the biomass of primary leaves while a significant reduction of first trifoliates dry matter could be observed (NF+60 atmosphere, third and fourth harvest). Among the normally occurring phenolics, we detected a significant decrease in the accumulation of a hydroxycinnamic acid derivative as the ozone concentration increased. Nevertheless, we demonstrated that this ozone-induced modification could be sometimes distinguishable with difficulties from changes expected to be of development relevance. Beside this phenolic disbalance, we detected a de novo biosynthesis of compounds that closely depended on the level of visible ozone injury. Since their accumulation increased with leaf damage, these ozone-induced phenolics could be used to detect phytotoxic ambient levels of tropospheric ozone.     

臭氧胁迫对水稻生长以及C、N、S元素分配的影响

采用开顶式气室(Open-top Chamber, OTC),对水稻"3694繁"(Oryza sativa L., 3694 Fan)在浙江嘉兴进行田间原位臭氧(O₃)熏气实验,研究不同臭氧浓度熏气对水稻生长以及C、N,S元素分配的影响。实验设置分4个水平:过滤大气组(CF,10 nL/L)、自然大气组(NF,40 nL/L)和两个不同浓度的臭氧处理组(O₃-1:100 nL/L; O₃-2:150 nL/L)。主要结果表明:(1)开始臭氧熏气时,各个处理组单茎水稻各组分生物量没有差异. 在熏气后期(水稻成熟期),臭氧处理使单茎水稻根、茎和穗生物量显著下降,根冠比降低,株高显著降低,表明臭氧胁迫增加水稻地上部分的干物质分配,且对株高的影响可能大于对地上生物量的影响;(2)臭氧处理使水稻根和茎C元素含量下降,叶C元素含量上升,表明臭氧胁迫提高了叶片中碳分配,而降低了根和茎的碳分配;(3)各个组分N元素含量上升和碳氮比下降;(4)茎、叶和穗S元素含量上升,可能会增强水稻抗氧化系统的作用,从而抵抗臭氧胁迫。所有实验结果表明臭氧浓度升高会对水稻生长产生严重不利影响,从而导致水稻各个组分的C、N、S元素分配格局发生改变。     

Metabolic-dependent changes in plant cell redox power after ozone exposure

The tropospheric level of the phytotoxic air pollutant ozone has increased considerably during the last century, and is expected to continue to rise. Long-term exposure of higher plants to low ozone concentrations affects biochemical processes prior to any visible symptoms of injury. The current critical level of ozone used to determine the threshold for damaging plants (biomass loss) is still based on the seasonal sum of the external concentration above 40 nl·l⁻¹ (AOT40). Taking into account stomatal conductance and the internal capacity of leaf defences, a more relevant concept should be based upon the 'effective ozone flux', the balance between the stomatal flux and the intensity of cellular detoxification. The large decrease in the Rubisco/PEPc ratio reflects photosynthetic damage from ozone, and a large increase in activity of cytosolic PEPc, which allows increased malate production. Although the direct detoxification of ozone (and ROS produced from its decomposition) is carried out primarily by cell wall ascorbate, the existing level of this antioxidant is not sufficient to indicate the degree of cell sensitivity. In order to regenerate ascorbate, NAD(P)H is needed as the primary supplier of reducing power. It is hypothesised that increased activity of the catabolic pathways and associated shunts (glucose-6-phosphate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malic enzyme) can provide sufficient NAD(P)H to maintain intracellular detoxification. Thus, measurement of the level of redox power would contribute to determination of the 'effective ozone dose', serving ultimately to improve the ozone risk index for higher plants.     

Rate and duration of grain filling in relation to flag leaf senescence and grain yield in spring wheat (Triticum aestivum) exposed to different concentrations of ozone

Effects of different concentrations of ozone on grain filling, flag leaf senescence and final grain yield in field‐grown spring wheat (Triticum aestivum L. cv. Dragon) were studied using open‐top chambers. The hypothesis tested was that an ozone‐induced reduction in grain yield is mainly related to an enhanced senescence and a shortening of the grain‐filling period. The plants were exposed to filtered air (F), non‐filtered air without extra ozone (NF) or non‐filtered air with 3 different levels of ozone added (NF1+, NF2+ and NF3+). The mean daytime (08.00–20.00 h) ozone concentrations during the exposure period (31 days) were 7, 20, 34, 48 and 62 nmol mol?1 in F, NF, NF1+, NF2+ and NF3+, respectively. The corresponding ozone doses, expressed as the accumulated exposure over a concentration threshold of 40 nmol mol?1 (AOT40), were 0, 12, 1 989, 5 881 and 10 375 nmol mol?1 h, respectively, and 884, 2 594, 4 557, 6 188 and 7 900 μmol m?2, respectively, expressed as the calculated cumulative flag leaf ozone flux (CFO30). The flag leaves senesced earlier and the grain‐filling duration was significantly shorter at higher ozone exposure compared to F (?5, ?13 and ?18% in NF1+, NF2+ and NF3+, respectively). The relative grain‐filling rate did not differ between the treatments. The 1000‐grain weights were 10, 28 and 37% lower, and the grain yields were 15, 29 and 46% lower than F in NF1+, NF2+ and NF3+, respectively. Ozone exposure had no significant effect on the number of grains per unit ground area or on straw yield, but significantly reduced the harvest index and increased the grain protein concentration in NF2+ and NF3+ compared to F. The grain yield was negatively correlated with the ozone dose, expressed either as AOT40 or as CFO3 with or without an ozone flux threshold. The 1000‐grain weight was positively correlated with the grain‐filling duration (R2=0.998), which in turn was positively correlated with the leaf area duration (R2=0.989).     

冬小麦气孔臭氧通量拟合及通量产量关系的比较分析

基于田间原位开顶箱(Open-Top Chambers,OTCs)实验,研究了不同浓度臭氧(O3)处理下(自然大气处理,AA;箱内大气处理,NF;箱内低浓度O3处理,NF+40 nL/L;箱内中等浓度O3处理,NF+80 nL/L;箱内高浓度O3处理,NF+120 nL/L),冬小麦(Triticum aestivum L.)旗叶气孔运动对不同环境因子的响应,并通过剂量反应分析,比较了冬小麦产量损失与累积气孔O3吸收通量(AFstX)和累积O3暴露浓度(AOT40和SUM06)的相关性差异。结果表明:冬小麦旗叶气孔运动的光饱和点和最适温度分别约为400μmol.m-.2s-1和27℃,水汽压差、土壤含水量和O3剂量的气孔限制临界值分别约为1.4 kPa、-100 kPa和20μL.L-.1h-1,超过此临界值时,气孔导度会明显下降。利用Jarvis气孔导度模型对冬小麦旗叶气孔导度和气孔O3吸收通量进行了预测,结果表明Jarvis模型解释了冬小麦实测气孔导度60%的变异性。由于不同时期植物体气孔导度的差异,冬小麦旗叶生长期内累积气孔O3吸收通量(AFstX)呈非线性增加趋势。O3吸收速率临界值(X)为4 nmol.m-.2s-1时,累积O3吸收通量(AFst4)与冬小麦产量的相关性最高(R2=0.76),该数值介于O3暴露指标AOT40和SUM06的剂量反应决定系数(0.74和0.81)之间。与O3浓度指标(AOT40和SUM06)相比,O3通量指标(AFstX)在本试验冬小麦产量损失评价中未表现出明显优势。     

Identification of phosphoenolpyruvate carboxylase isoforms in leaf, stem and roots of the obligate CAM plant Vanilla planifolia Salib. (Orchidaceae): a physiological and molecular approach

This study provides the first comparative analysis of phosphoenolpyruvate carboxylase isoforms (PEPc; EC 4.1.1.31) in an obligate crassulacean acid metabolism (CAM) plant, Vanilla planifolia Salisb. (Orchidaceae). Nocturnal CO2 fixation and malate accumulation by the leaves and the green stem show that these organs perform CAM. The chloroplast-containing aerial roots, however, exhibit C3 photosynthesis. The catalytic activity of PEPc was highest in the leaves compared with the stem and aerial roots. The Km (PEP) and Ki (malate) were similar in the PEPc extracted from leaf and aerial roots, and significant higher in stem. cDNA was obtained from those tissues and also from the soil-grown roots, and various cDNA clones were detected and amplified by means of RT-PCR and RACE-PCR. The amino-acid sequences of the PEPc isoforms deduced from the cDNA showed a great degree of homology, and Southern blot analysis suggests that the encoding genes form a small multigene family of at least two members. One PEPc isoform (PpcV1) is assumed to be related to CAM because, as shown by northern blot analysis, it is mainly expressed in the CAM-performing organs, i.e. in the leaves and the stem. A further isoform (PpcV2) was identified in the soil-grown roots and aerial roots, but northern blots show that to some extent PpcV2 is also expressed in the leaf and the stem tissues. Thus, it is assumed that PpcV2 encodes the housekeeping isoform of PEPc. Altogether, the present study provides support in favour of the view that isoforms of PEPc are related to specific functions.     

Survival and recovery of perennial forage grasses under prolonged Mediterranean drought: II. Water status, solute accumulation, abscisic acid concentration and accumulation of dehydrin transcripts in bases of immature leaves

Effects of ozone on spring wheat ( Triticum aestivum L. cv. Satu) were studied in an open-top chamber experiment during two growing seasons (1992–1993) at Jokioinen in south-west Finland. The wheat was exposed to filtered air (CF), non-filtered air (NF), non-filtered air+35 nl l⁻¹ ozone for 8 h d⁻¹ (NF⁺) and ambient air (AA). Each treatment was replicated five times. Two wk after anthesis, after 4 wk of ozone treatment (NF⁺, 45 nl l⁻¹ 1000–1800 hours, seasonal mean) the net CO₂ uptake of wheat flag leaves was decreased by c . 40% relative to CF and NF treatments, both initial and total activity of Rubisco and the quantity of protein-bound SH groups were decreased significantly. Added ozone also significantly accelerated flag leaf senescence recorded as a decrease in chloroplast size. The effect was significant 2 wk after anthesis, and senescence was complete after 4 wk. In the CF and NF treatments senescence was complete 5 wk after anthesis. The significant effect of ozone on the chloroplasts and net CO₂ uptake 2 wk after anthesis did not affect the grain filling rate. However, since the grain filling period was shorter for ozone fumigated plants, kernels were smaller. The decrease in 1000-grain weight explained most of the yield reduction in the plants under NF⁺ treatment. The results indicate that wheat plants are well buffered against substantial decrease in source activity, and that shortened flag leaf duration is the major factor causing ozone-induced yield loss.     

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.     

Competing carboxylases: circadian and metabolic regulation of Rubisco in C3 and CAM Mesembryanthemum crystallinum L

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL.     

Biomass, reproductive output, and physiological responses of rapid-cycling Brassica (Brassica rapa) to ozone and modified root temperature

Brassica rapa L. (rapid-cycling Brassica), was grown in environmentally controlled chambers to determine the interactive effects of ozone (O₃) and increased root temperature (RT) on biomass, reproductive output, and photosynthesis. Plants were grown with or without an average treatment of 63 ppb O₃. RT treatments were 13°C (LRT) and 18°C (HRT). Air temperatures were 25°C/15°C day/night for all RT treatments.
Ozone affected plant biomass more than did root temperature. Plants in O₃ had significantly smaller total plant d. wt, shoot weight, leaf weight, leaf area and leaf number than plants grown without O₃. LRT plants tended to have slightly smaller total plant d. wt, shoot weight, root weight, leaf weight, leaf area, and leaf number than HRT plants. For all variables, LRT plants grown in O₃ had the smallest biomass, and plants grown in HRT without O₃ had the largest biomass.
Ozone reduced both fruit weight and fruit number; LRT also reduced fruit weight but had no effect on fruit number. Ozone reduced photosynthesis but RT had no effect. Conductance and internal CO₂ were unaffected by O₃ or RT.
These studies indicate that plant growth with LRT might be more reduced in the presence of O₃ than growth in plants with HRT, which might be able to compensate for O₃-caused reductions in photosynthesis to avoid decreased biomass and reproductive output.     

Growth and photosynthesis under high and low irradiance of Arabidopsis thaliana antisense mutants with reduced ribulose-1,5-bisphosphate carboxylase/oxygenase activase content.

Photosynthesis and growth to maturity of antisense ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase Arabidopsis thaliana with reduced concentrations of activase relative to wild-type (Wt) plants were measured under low (200 mumol m-2 s-1) and high (600 mumol m-2 s-1) photosynthetic photon flux density growing conditions. Both growth and photosynthesis were significantly reduced in an Arabidopsis clone (R100) with 30 to 40% Wt activase, an effect that was more pronounced in high light. The aboveground biomass of the antisense clone R100 reached 80% of Wt under low light and 65% of Wt under high light. Decreased growth in the antisense plants was attributed to reduced relative rates of growth and leaf area expansion early in development; all plants attained similar values of relative rates of growth and leaf elongation by 21 d after planting. Reductions in photosynthesis were attributed to decreased Rubisco activation in the antisense plants. Rubisco constituted about 40% of total soluble protein in both Wt and clone R100 under both light regimes. Activase content was 5% and 1.4% of total soluble protein in Wt and clone R100, respectively, and also was unaffected by growth irradiance. The stoichiometry of Rubisco to activase was estimated at 20 Rubisco active sites per activase tetramer in Wt Arabidopsis and 60 to 80 in the transgenic clone R100. We conclude that Wt Arabidopsis does not contain Rubisco activase in great excess of the amount required for optimal growth.     

Differential expression of photosynthesis genes in leaf tissue layers of Peperomia as revealed by tissue printing

Peperomia has species that may be C₃, show Crassulacean acid metabolism (CAM), or CAM-cycling. Species that show CAM progress from C₃ to CAM through CAM-cycling during leaf development. In CAM and CAM-cycling species, CAM metabolism is predominately in the upper multiple epidermis and lower spongy mesophyll, whereas C₃ metabolism is localized mostly in the palisade mesophyll. Using specific protein and cDNA probes prepared from P-enolpyruvate carboxylase (PEPc) and ribulose-1,5-bisphosphate carboxylase (Rubisco), we have now studied the differential distribution of photosynthetic metabolism in Peperomia leaves using the technique of tissue printing. The tissue printing studies detected Rubisco protein in leaves of C₃ P. orba, but not PEPc. Young C₃ leaves of P. scandens and P. camptotricha showed Rubisco protein, but not PEPc; however, the mature leaves of these two species that have CAM showed PEPc protein and RNAs in both the multiple epidermis and spongy mesophyll. In contrast, Rubisco protein and RNAs were present throughout the leaf. The tissue printing data are consistent with our previously published data showing the differential distribution of photosynthetic metabolism in leaves of Peperomia. Although the tissue printing technique is qualitative, coupled with quantitative data it has proven useful for the study of function related to structure.     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).     

Expression of P-Enolpyruvate Carboxylase and other Aspects of CAM during the Development of Peperomia camptotricha Leaves

During the development of Peperomia camptotricha leaves, metabolism changes from C₃-photosynthesis to Crassulacean acid metabolism (CAM). The youngest leaves showed no diurnal fluctuation of organic acids or P-enolpyruvate carboxylase (PEPc) activity. There was little evidence for PEPc protein using PEPc antibodies prepared from the CAM form of PEPc, nor was there evidence for PEPc mRNA when tested using a cDNA probe made from CAM P. scandens. As leaves matured, there was a parallel increase in titratable acidity, PEPc activity, PEPc protein, and PEPc mRNA. In leaf whorls 1 through 6, there was a significant linear correlation between the diurnal fluctuation of organic acids and PEPc activity indicating a functional relationship. The specific activity of PEPc increased as leaves matured and the K_m (PEP) decreased indicating that the enzyme was becoming more active. The ratio of PEPc protein to PEPc mRNA decreased as leaves matured. During the expression of CAM, the spongy mesophyll where most of the CAM activity occurs increased in thickness and per cent air space, whereas the palisade mesophyll where most of the C₃ activity occurs did not increase in size dramatically. The diurnal fluctuation of organic acids and the expression of PEPc activity, protein, and mRNA increased as the thickness of the spongy mesophyll increased. During the expression of CAM in Peperomia camptotricha, there appears to be coordinated expression of PEPc mRNA, protein, and activity, the commencement of diurnal organic acid fluctuation, and the development of the CAM-like spongy mesophyll. Thus the evidence suggests that CAM in this species is expressed during normal development and not in response to environmental signals.     

Raman spectroscopy of pigments and oxalates in situ within epilithic lichens: Acarospora from the Antarctic and Mediterranean

The effects of ambient and elevated ozone levels on growth and photosynthesis of beech ( Fagus sylvatica ) were studied by exposing seedlings in open-top chambers for one growing season to three treatments: charcoal-filtered (CF), non-filtered (NF) and non-filtered air with addition of ozone (30 ppb ozone) on clear days for 8–10 h d⁻¹ (NF +). Ambient levels were relatively low and accumulated to an AOT40 (accumulated exposure over a threshold of 40 ppb) of 4055 ppb h (for the period 23 Apr–30 Sept). The NF + chambers received an AOT40 of 8880 ppb h. Throughout the growing season we measured growth and photosynthetic properties. The treatments did not cause strong effects: measurements of gas exchange (light-saturated assimilation rate, CO₂ and light-response curves) and chlorophyll fluorescence showed slight and mostly non-significant reductions of several parameters. No significant differences were found for growth, though in the NF + treatment (AOT40 8880 ppb h) the relative growth rate for diameter increment was at times reduced by 12% compared with the control treatment.     

Growth parameters and resistance against Drechslera teres of spring barley (Hordeum vulgare L. cv. Scarlett) grown at elevated ozone and carbon dioxide concentrations

Spring barley ( Hordeum vulgare L. cv. Scarlett) was grown at two CO2 levels (400 vs. 700 ppm) combined with two ozone regimes (ambient vs. double ambient) in climate chambers for four weeks, beginning at seedling emergence. Elevated CO2 concentration significantly increased aboveground biomass, root biomass, and tiller number, whereas double ambient ozone significantly decreased these parameters. These ozone-induced reductions in growth parameters were strongly overridden by 700 ppm CO2. The elevated CO2 level increased C : N ratio of the leaf tissue and leaf starch content but decreased leaf protein levels. Exposure to double ambient ozone did not affect protein content and C : N ratio but dramatically increased leaf starch levels at 700 ppm CO2. Resistance against Drechslera teres (Sacc.) Shoemaker was increased in leaves grown at double ambient ozone but was less obvious at 700 ppm than at 400 ppm CO2. Constitutive activities of beta-1,3-glucanase and chitinase were significantly higher in leaves grown at double ambient ozone compared to ambient ozone levels. The sum of methanol-soluble and alkali-released cell wall-bound aromatic metabolites (i.e., C-glycosylflavones and several structurally unidentified metabolites) and lignin contents did not show any treatment-dependent differences.